Effects Of BENC-511 On Proliferation And Apoptosis In Human Lung Adenocarcinoma A549 Cells

Lung cancer is one of the clinical common malignant tumors, which stands first on the list of deadly tumor diseases in the world and threat to people’s health and life. Nearly 50 years, the morbidity and mortality of lung cancer are significantly increased. Among several types of lung cancer, the incidence of non-small cell lung cancer (NSCLC) has been rising in recent years and has become the most common type of lung cancer. NSCLC includes squamous cell carcinoma (cancer), adenocarcinoma and large cell carcinoma, and is easy to transfer and results to drug resistance with the characteristics of high mortality and morbidity Compared with small cell carcinoma, NSCLC cells divide more slowly and spread relatively late. NSCLC accounted for about 80% of all lung cancer, about 75% of the patients has been found in the middle-late phage,5-year survival rate is very low.For the treatment of lung cancer, the clinical commonly used chemotherapy drugs mainly direct effects on cell mitosis, DNA synthesis and repair process, such as traditional cytotoxic drugs, these drugs have low selectivity and high toxicity. Therefore, the key enzyme of related to tumor cell differentiation and proliferation signal pathways for drug screening targets, looking for selective effects on specific targets for new anti-cancer drugs has become the important direction of antitumor drug development today. And these small molecule inhibitors have better curative effects and less side effects compared with traditional cancer chemotherapy drugs. Phosphatidyl inositol-3-kinase (PI3K), one of the lipid kinase, is the key signal molecules in cell life activities and exist in all kinds of cells in the body. Through the recruitment and activation of downstream target material, launched a series of cascade signal, PI3K/Akt plays an important role in the process of cell growth, proliferation, survival, differentiation, apoptosis, and metabolism, and other many kinds of cell biology. Studies have shown that PI3K/Akt signaling pathway is closely related to the occurrence of tumor development. Key enzymes in the signal pathway as target of anti-tumor drug research become one of the hot spots in tumor prevention and tumor therapy.Compound S14161,8-ethoxy-2-(4-fluorinated phenyl)-3-hydrogen nitro-2-benzo pyran, has been identified as an inhibitor of PI3K and could inhibit tumor growth. But the chiral structure and poor solubility prevent its further application. In order to obtain more active compounds, Professor Zhaopeng Liu in our institute of pharmaceutical sciences designed a novel analogue of S14161 through structural optimization, 6-bromo-8-ethoxy-3-nitro-2H-chromene (BENC-511), which exhibited potent antiproliferative activities. Preliminary experiments showed that BENC-511 had good effects on multiple myeloma and low toxicity by inhibiting PI3K/Akt pathway, Akt phosphorylation level and the expression of downstream signaling proteins. But the effects of BENC-511 on human lung adenocarcinoma A549 cells are not reported.In the present study, effects of BENC-511 on proliferation and apoptosis in A549 cells in vitro were investigated. The inhibitory effects of BENC-511 on proliferation of human lung adenocarcinoma A549 cell were determined by MTT assay and clone formation test after treated with BENC-511. Light inverted microscope was used to observe the morphology change of A549 cells. The effects of BENC-511 on A549 cell cycle and DNA content were detected by Flow cytometry, the expression of CyclinA, PCNA(proliferating cell nuclear antigen, PCNA), p21, Caspase-3, Caspase-9, and P-Akt was measured through Western blotting; The effects of BENC-511 on apoptosis of A549 cells were detected by flow cytometry using Annexin V-FITC/PI double staining. The mitochondrial membrane potential (△Ψm) were measured by JC-1 reagent kit in fluorescence microscopy.After exposure to different concentrations of BENC-511 for 24,48 and 72h, the cell viability of A549 cells was inhibited in a concentration-and time-dependent manner, consistent with the results of clone formation inhibition test. Changes in cell morphology were observed by light microscope. Results showed that the cell morphology of A549 treated by 1.25,2.5,5 μmol/L BENC-511 changed significantly. Cell density decreased significantly, meanwhile the cells gradually shrank, turned round, and appeared apoptotic body. Flow cytometry results showed that, after treated by BENC-511, the proportion of A549 cells in S phase cells increased significantly, which suggested that the cell cycle was arrested in S phase. The number of Annexin V positive cells increased in a concentration dependent manner. Compared with S14161,10 μmol/L BENC-511 induced apoptosis of A549 cells more obviously. Western blot results showed that BENC-511 could decrease the expression of cyclin A1,PCNA and P-Akt, increase the expression of p21, Caspase-3 and Caspase-9. JC-1 detection of cell mitochondrial membrane potential showed that the JC-1 gradually changed from the red fluorescence to the green fluorescence which indicated that mitochondrial membrane potential decreased.In conclusion, BENC-511 inhibited human lung adenocarcinoma A549 cells proliferation and induced apoptosis by influencing cycle and reducing the associated cells mitochondrial membrane potential, the detailed mechanism still needs further research.